Research Plan: Our studies have established that impaired alveolar epithelial fluid clearance is associated with a higher mortality in patients with acute lung injury. During the first cycle of this award, we published several studies that markedly increased our understanding of the mechanisms that impair alveolar epithelial function in clinical acute lung injury (see Progress Report). Additional studies are needed to increase our understanding of the biological mechanisms that impair alveolar epithelial fluid clearance in clinical lung injury and to test therapeutic strategies that may increase the clearance of alveolar edema fluid and accelerate the resolution of lung injury. In Aim 1, we will use pulmonary edema fluid and plasma from critically ill patients with acute lung injury to study the mechanisms that mediate an increase in protein permeability and a decrease in vectorial fluid transport across cultured human alveolar epithelial type 11 cells that form tight monolayers grown in an air-liquid interface. Controls will be done with edema fluid and plasma from patients with cardiogenic pulmonary edema. In Aim 2, we will collect samples of pulmonary edema fluid and plasma from patients with early acute lung injury to (1) measure alveolar fluid clearance, (2) biological markers of lung epithelial and endothelial injury, and (3) clinical measurements of lung function to assess the biologic and physiologic mechanisms that contribute to impaired alveolar fluid clearance in clinical lung injury. In Aim 3, we will use our novel preparation of perfused human lungs harvested from brain dead donors to measure lung endothelial and epithelial permeability and alveolar epithelial fluid clearance in order to test the effect of clinically relevant therapeutic strategies (very low tidal volume (3 ml/kg), high frequency ventilation, dopamine, interleukin-1 receptor antagonist, and growth factors) on lung fluid balance over 4-8 hours. In summary, the integrated studies of biochemical, physiological and clinical measurements in plasma and pulmonary edema fluid in critically ill patients in Aim 2 will generate new insights into the mechanisms that impair alveolar epithelial fluid clearance in clinical acute lung injury. The studies in Aims 1 and 3 studies will provide innovative, novel approaches that use human alveolar type II cells and human lungs to study the mechanisms of alveolar epithelial injury as well as to test the potential value of several clinically relevant therapeutic strategies in pre-clinical studies.